Physiologically based Kinetic Modeling-Facilitated Quantitative In Vitro to In Vivo Extrapolation to Predict the Effects of Aloe-Emodin in Rats and Humans

Aloe-emodin, a natural hydroxyanthraquinone, exerts both adverse and protective effects. This study aimed at investigating these potential effects of aloe-emodin in humans upon the use of food supplements and herbal medicines using a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach. For this, PBK models in rats and humans were established for aloe-emodin including its active metabolite rhein and used to convert in vitro data on hepatotoxicity, nephrotoxicity, reactive oxidative species (ROS) generation, and Nrf2 induction to corresponding in vivo dose–response curves, from which points of departure (PODs) were derived by BMD analysis. The derived PODs were subsequently compared to the estimated daily intakes (EDIs) resulting from the use of food supplements or herbal medicines. It is concluded that the dose levels of aloe-emodin from food supplements or herbal medicines are unlikely to induce toxicity, ROS generation, or Nrf2 activation in liver and kidney.


Quantification of aloe-emodin and its metabolite rhein by LC-MS/MS
The quantification of aloe-emodin and rhein was carried out by LC-MS/MS analysis with a Phenomenex Kinetex C18 column (1.7 µm, 2.1 × 50 mm; Phenomenex, Utrecht, The Netherland).The mobile phase A was water containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid.The flow rate was 0.

Quantification of aloe-emodin and its glucuronides by UPLC-PDA
To quantify the concentration of aloe-emodin and its glucuronides, a UPLC Nexera series (Shimadzu, Kyoto, Japan) equipped with a Photodiode Array (PDA) detector was utilized.Aloe-emodin and its glucuronides were separated on a Phenomenex C18 column (50  2.1 mm, 1.7 µm).Mobile phase A was nanopure water with 0.1% TFA and mobile phase B was acetonitrile.The gradient was: 10-90% B for 0-9.00 min; 90% B for 9.00-10.00min, 90%-10% B for 10.00-10.30min; 10% B for 10.30-15.00min.5 µl of sample were loaded on the column and elution was performed with a flow rate of 0.3 ml/min.The temperature of the column was at 40 C.At a wavelength of 225 nm, aloe-emodin and its glucuronides were quantified based on comparison of the respective peak areas to the peak areas of a linear calibration curve for aloe-emodin prepared in Tris-HCl pH 7.4 containing 20% (v/v) ACN reference standards.

Calculation of kinetic parameters
The conversion of aloe-emodin to rhein and the formation of glucuronidation from aloe-emodin in in vitro incubations with liver microsomes and liver S9 fractions were fitted to a standard Michaelis-Menten equation (Eq S1) to calculate kinetic parameters: (Eq S1) in which [S] is the substrate concentration (µM), v is the rate of metabolite formation (nmol/min/mg microsomal protein or nmol/min/mg S9 protein), Vmax is the apparent maximum reaction rate (nmol/min/mg microsomal protein or nmol/min/mg S9 protein), along with an Michaelis-Menten constant (Km, in µM).
To quantify the in vitro clearance rate (CLint,in vitro) for rhein, the remaining concentration of rhein in the incubations of rhein with rat hepatocytes (Crhein) was compared to that in the corresponding incubation of rhein without rat hepatocytes as control sample (Ccontrol) at each incubation time point.This allowed to derive the depletion curve of rhein against time, which was expressed as [ln (Crhein / Ccontrol)].The slope of the linear portion of the depletion curve represents the depletion rate constant (k, in min -1 ), and the CLint, in vitro was calculated using the following equation (Eq S2): CLint,   (ml/min/million cells) =  (min −1 )× () ( ) (Eq S2) In which, k represents the depletion rate constant (min -1 ), V the incubation volume (0.2 mL), and n the number of cells in the incubation (0.5 million cells).
As stated in section 3.2 in manuscript, the in vitro clearance of rhein with primary human hepatocytes was taken from literature where it was reported to be negligible amounting to 0 μL/min/million cells 1 .

Sensitivity analysis
A local sensitivity analysis was conducted to assess the influence of individual parameters on the output of maximum concentration values of aloe-emodin or rhein from the PBK model.The normalized sensitivity coefficient (SC) was calculated with the following equation (Eq S3): In which C is the initial value of the model output, C' is the modified value of the model output with a 5% increase of an input parameter; P is the initial parameter value and P' is the 5% increased input parameter value.Only one parameter was changed at a time.A parameter with an absolute value of SC greater than 0.1 is considered as an influential parameter on the output of the PBK modeling 2,3 .The larger the absolute value of SC, the higher the influence of the parameter on the model output.The sensitivity analysis was performed for Cmax as the parameter of interest and for a single oral administration of 40 or 300 mg/kg BW aloe-emodin, dose levels used in reported in vivo studies 4,5 .
Figure S2.In vitro concentration-response curves for nephrotoxicity of aloe-emodin in kidney cells as reported in literature 10 .
Note: Figure S8 shows the parameters with a normalized sensitivity coefficients higher than 0.1 obtained at 5% increase compared to their respective original values after oral administration of aloe-emodin at a single oral dose of 40 mg/kg BW in rat, a dose equal to one of the dose levels used in the reported in vivo studies 5 and a single oral dose of 0.066 mg/kg BW in human which is the geometric mean of daily exposure to aloe-emodin from medical use derived from reported data (Table S1).The results show that for both aloe-emodin and rhein in rat, fraction of liver (VLc), ka, Fa, liver S9 protein yield (VLS9), and the Vmax and Km for formation of AEG1 and AEG2 are the most influential parameters for prediction of blood concentrations of aloe-emodin and rhein while liver microsomal protein yield (MPL), and the Vmax and Km for conversion of aloe-emodin to rhein, CLinthepRH and hepatocytes number in liver (CellDL) have more influence on the predicted Cmax values for rhein than for aloe-emodin.In the human model, ka and Fa have substantial effects on the Cmax for both aloe-emodin and rhein.VLc shows a higher influence on the predicted Cmax for aloe-emodin while GF, MPL, Vmax and Km for rhein show a higher influence on the predicted Cmax for rhein.

Figure S4 .
Figure S4.Overlay of HPLC-UV chromatograms of incubations of aloe-emodin with (A) rat liver S9 or (B) human liver S9 with (red) or without (black) beta-glucuronidase treatment.

Figure
Figure S5.PBK modeling-based predictions of dose-dependent maximum concentration of aloe-emodin in venous blood of liver and venous blood of kidney used for QIVIVE.

Figure S6 .
Figure S6.Comparison of time-dependent predicted blood concentration of aloe-emodin and rhein with reported in vivo data after a single (A. and C. 40 mg/kg 4 ; B. 300 mg/kg 5 ) oral administration of aloe-emodin in rats (ka = 0.21 h -1 ; Fa = 0.26).Enterohepatic circulation was not included in the PBK model explaining part of the deviations.For further details see text in manuscript.

Figure S7 .
Figure S7.Comparison of time-dependent predicted blood concentration of aloe-emodin and rhein with reported in vivo data after a single (A. and C. 40 mg/kg 4 ; B. 300 mg/kg 5 ) oral administration of aloe-emodin in rats (ka = 4 h -1 ; Fa = 0.022).Enterohepatic circulation was not included in the PBK model explaining part of the deviations.For further details see text in manuscript.

Figure S8 .
Figure S8.Local sensitivity analysis for the predicted maximum blood concentration of aloe-emodin and rhein in (A) rats and (B) humans after oral administration of aloe-emodin at a single dose of 40 mg/kg BW (rats) and 0.066 mg/kg BW (humans).Only model parameters with a normalized SC higher than 0.1 (absolute value) are shown.The parameters represent: VLc: fraction of liver tissue; VSc: fraction of slowly perfused tissue; QLc: fraction of blood flow to liver; PSAE: slowly perfused tissue:blood partition coefficient of aloe-emodin; PSRH: slowly perfused tissue:blood partition coefficient of rhein; ka: absorption rate constant of aloe-emodin from intestine to liver; Fa: fraction of dose absorbed; kb: bile excretion constant; GF: glomerular filtration rate; MPL: liver microsomal protein yield; VLS9: liver S9 protein yield; VmaxRHc maximum rate for conversion of aloe-emodin to rhein; KmRH: Michaelis-Menten constant for conversion of aloe-emodin to rhein; VmaxAG1c: maximum rate for conversion of aloe-emodin to AEG1;

Table S2 .
Physiochemical parameters of aloe-emodin and rhein in rats and humans, determined as described in Materials and Methods.

Table S3 .
Botanical source, content in food supplements, its recommended daily use dosage by suppliers, content of aloe-emodin in the botanicals and the estimated daily intakes of aloe-emodin from food supplements use.

Table S4 .
The botanical daily intake, the content of aloe-emodin in the botanicals and the estimated daily intakes of aloe-emodin from medicinal use.
a botanical daily intake was from Chinese Pharmacopoeia 2020 edition.b EDIs were calculated by "(Botanical daily intake as recommended by the Chinese Pharmacopoeia (2020 edition)  Content of aloe-emodin)/Body weight (60 kg)".

Table S6 .
Summary of in vitro data of aloe-emodin in cell models and corresponding predicted BMDL10 and BMDU10 values obtained by using PBK modeling-facilitated QIVIVE.